Genetic and morphologic variation in the Davis Mountains cottontail (Sylvilagus robustus).
Abstract: The Davis Mountains cottontail, Sylvilagus robustus, is an endemic species of the Trans-Pecos area of Texas. Although S. robustus previously was believed to be extirpated from the Chisos Mountains, we confirmed existence of a population. We examined intrapopulation and interpopulation variation in S. robustus, as well as the genetic relationship to the eastern cottontail (S. floridanus) using partial sequences of the mitochondrial cytochrome b and control region genes. Six morphometric traits relating to overall size and six cranial characters considered diagnostic for the two subspecies were used to confirm identification of specimens. Our morphological analysis suggested that specimens from the Chisos and Davis Mountains were S. robustus; however, low levels of genetic divergence between S. robustus and S. floridanus appeared inconsistent with species-level recognition for S. robustus.

El conejo del monte, Sylvilagus robustus, es una especie endemica de la zona de Trans-Pecos de Texas. Aunque S. robustus fue anteriormente considerada erradicada de las montarias Chisos, confirmamos la existencia de una poblacion. Examinamos la variacion genetica dentro y entre poblaciones en S. robustus, y tambien la relacion genetica con el conejo de castilla (S. floridanus) usando secuencias parciales de genes de ADN mitocondriales de la region de control y de citocromo b. Seis caracteristicas morfometricas relacionadas con el tamano corporal y seis caracteristicas de craneos consideradas diagnosticas para las dos subespecies fueron usadas para confirmar la identificacion de los especimenes. Nuestro analisis morfologico sugirio que los especimenes colectados de las montanas Chisos y Davis fueron S. robustus; sin embargo, los bajos niveles de divergencia genetica entre S. robustus y S. floridanus parecieron inconsistentes con el reconocimiento del nivel de especie para S. robustus.
Article Type: Report
Subject: Cottontails (Genetic aspects)
Genetic variation (Identification and classification)
Morphology (Animals) (Research)
Authors: Nalls, Amy V.
Ammerman, Loren K.
Dowler, Robert C.
Pub Date: 03/01/2012
Publication: Name: Southwestern Naturalist Publisher: Southwestern Association of Naturalists Audience: Academic Format: Magazine/Journal Subject: Biological sciences Copyright: COPYRIGHT 2012 Southwestern Association of Naturalists ISSN: 0038-4909
Issue: Date: March, 2012 Source Volume: 57 Source Issue: 1
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Name: Davis Mountains Geographic Code: 1USA United States
Accession Number: 285321077
Full Text: The Davis Mountains cottontail, Sylvilagus robustus, is an endemic species of the Trans-Pecos region of Texas, occurring in pinyon-oak-juniper (Pinus-Quercus-Juniperus) woodlands of the Guadalupe, Davis, Chinati, and Chisos Mountains at elevations of 1,432-2,438 m (Schmidly, 1977). Sylvilagus robustus is larger and darker than the widely distributed eastern cottontail, S. floridanus (Schmidly, 1977). Specimens also are known from Coahuila, Mexico, originally described as S. floridanus nelsoni by Baker (1955) but synonymized with S. robustus by Raun (1965). Reports, but no specimen, of this cottontail were obtained in the Sierra del Carmen of northern Mexico (Baker, 1956).

Sylvilagus robustus was first described as Lepus pinetis robustus from specimens collected by V. Bailey at an elevation of 1,829 m in the Fort Davis area of Jeff Davis County, Texas (Bailey, 1905). Sylvilagus robustus was elevated to species level by Nelson (1909) on the basis of morphological features and lack of apparent intergrades, and it maintained this status until Hall and Kelson (1951) relegated the taxon to a subspecies of S. floridanus based on intermediate morphology of one specimen (Louisiana State University Museum of Natural Science 658) between S. f. cognatus and S. robustus. Davis (1974) gave an account of this taxon as S. robustus. Schmidly (1977) considered it a subspecies after examining specimens of S. floridanus from throughout Texas and New Mexico and observing only subspecific-level differences in robustus. Subsequently, Davis and Schmidly (1994:92) cited "nominal cranial differences with S. floridanus' as the basis for their subspecific designation of robustus. However, Ruedas (1998) conducted a multivariate morphological analysis of 26 cranial, mandibular, and dental characters examining five taxa of cottontails (S. floridanus chapmani, S. f. cognatus, S. f. holzneri, S. f. robustus, and S. nuttallii pinetis) and detected a separation of group means for six cranial characters between robustus and all other taxa examined (robustus having the largest means). Ruedas (1998) also reported differences in discrete cranial and mandibular characters in specimens of robustus and the parapatric S. floridanus chapmani. Schmidly (2004) followed this taxonomy, recognizing S. robustus as a species.

Collection of Davis Mountains cottontails has proven difficult in the past because of small populations that are presumably a result of low precipitation and productivity in the montane areas they inhabit (Ruedas, 1998). Prior to this study, no specimen was known from the Chisos or Guadalupe Mountains within the past 30 years (Ruedas, 1998). Schmidly (2004) reported a recent collection of several specimens from the Davis Mountains indicating that a healthy population remained there.

[FIGURE 1 OMITTED]

There currently are no published mitochondrial data examining S. robustus as a taxon, or its relationship to the parapatric S. floridanus chapmani, but morphological differences suggest some level of separation from S. floridanus. We examined variation in sequences across portions of the control region and cytochrome b gene in the mitochondrial-DNA genome to determine genetic relationships between S. robustus and S. floridanus, levels of gene flow between isolated populations of S. robustus, and genetic variation within populations. We also recorded morphological data for S. robustus and S. floridanus and compared these to results of Ruedas (1998).

MATERIALS AND METHODS--We sampled cottontails in the Chisos Mountains of Big Bend National Park, Brewster County, Texas, at elevations of 1,470-2,027 m during May 2003-August 2004 and in Lincoln National Forest, Guadalupe Mountains, Eddy County, New Mexico, at elevations of 1,806-2,164 m during October 2003-January 2004. We trapped for a total of 485 trapnights (one trapnight equals one trap set for one night) in Big Bend National Park and 154 trapnights in the Guadalupe Mountains. Specimens of Sylvilagus that were dead on the road in Big Bend National Park were salvaged if they were above 1,220 m elevation. We baited live traps with apples and alfalfa, as suggested by Forys and Humphrey (1997). We identified specimens as S. robustus if they were collected above the known lower-elevational limit for the species (1,432 m), and external measurements were consistent with those reported for the species (Schmidly, 2004). Samples of heart, liver, kidney, and muscle tissues were taken (when present) from each individual collected from Big Bend National Park and frozen for preservation. Voucher specimens and tissues were deposited in the Angelo State Natural History Collections (ASNHC). We included additional specimens from the Davis Mountains and southern New Mexico (Appendix) deposited in the collections at Eastern New Mexico University, Texas Tech University, and the Angelo State Natural History Collections.

We isolated total genomic DNA from liver, heart, muscle, or lung tissues using a DNeasy Tissue Kit (QIAGEN, Inc., Valencia, California) following protocol of the manufacturer. We amplified partial sequences of cytochrome b using universal primers L14841 (Kocher et al., 1989) and H15547 (Edwards et al., 1991), whereas partial sequences of the control region were generated using primers designed for chiropterans (e.g., CRP-L and CRFH; Wilkinson and Chapman, 1991).

Samples were amplified using an initial denaturing period at 93[degrees]C for 3 min, followed by 39 cycles of amplification (denaturation at 94[degrees]C, annealing at 48[degrees]C, and extension at 72[degrees]C for 1 min each), followed by a final extension period at 72[degrees]C for 3 min. PCR products were purified using low-melt agarose gels (1.0% w/v) premixed with 0.05% (10 ug/mL) ethidium bromide, then cloned using the TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, California) following protocol of the manufacturer (modified such that only one-fourth of the recommended volume was used). Plasmids containing PCR product were isolated with QIAprep Spin Miniprep Kit (QIAGEN, Inc., Valencia, California) and DNA sequenced using USB ThermoSequenase Cycle Sequencing Kit (United States Biochemical Corp., Cleveland, Ohio) and fluorescent dye-labeled primers (M13 forward [-29] IRDye 700 and M13 Reverse IRDye 800; Licor Biosciences, Lincoln, Nebraska). Sequences were analyzed on a Licor NEN Global IR2 DNA analyzer system using eSeq software (versions 2.0 and 3.0). We aligned sequences using the program Sequencher 4.1 (Gene Codes Corporation, Ann Arbor, Michigan) and compared to the following sequences of cottontails obtained from GenBank: S. floridanus mallurus (AY292724), S. audubonii (AY292722), and S. aquaticus (AY292726). Sylvilagus audubonii was used as the outgroup because it occupies a basal position to S. floridanus in the phylogeny of Sylvilagus (Halanych and Robinson, 1997). Sylvilagus aquaticus was included for additional comparison of specific-level, genetic distance.

The best-fit model of evolution of sequences of DNA for likelihood analysis was determined using MODELTEST version 3.06 (Posada and Crandall, 1998). Standard phylogenetic analyses using the maximum-likelihood method (Felsenstein, 1985) were conducted using [PAUP.sup.*] version 4.0b10 (Swofford, 2001). Statistical support of specific nodes was determined through likelihood bootstrap analyses (Felsenstein, 1985) using 100 replicates. Branches with bootstrap proportions >70% were considered well-supported (Hillis and Bull, 1993). For cytochrome b data, we calculated pair-wise divergence of sequences under the Kimura two-parameter model of evolution (Kimura, 1980) to facilitate comparison with divergences between pairs of species as in Bradley and Baker (2001).

We took actual measurements (when the carcass was intact) or used data from labels of museum specimens including total length, length of tail, length of hind foot, and length of ear as described by Schmidly (2004), as well as mass and gender for adult S. robustus and S. floridanus. We also measured six cranial characters reported by Ruedas (1998) to separate robustus from five other taxa of cottontails (i.e., greatest length of skull, condylopremaxillary length, breadth of rostrum, interbasioccipital length, width of auditory bullae, and mastoid breadth). All measurements were taken to the nearest 0.1 mm using digital calipers. We calculated mean, standard deviation, and range for robustus and chapmani and compared our results to data in Ruedas (1998). We also scored discrete mandibular and cranial characters identified by Ruedas (1998) to distinguish robustus and chapmani, including the basisphenoid foramina, tympanic process, and mental foramen.

RESULTS--Ten road-killed individuals were salvaged from within Big Bend National Park at elevations of 1,463-1,768 m, and only one was captured in a trap (ASNHC 12940). Collecting trips in the Guadalupe Mountains were unsuccessful. A list of individuals and collecting localities is provided in the Appendix.

We sequenced a total of 648 base pairs of the cytochrome-b gene for eight individuals from the Chisos Mountains, three from the Davis Mountains, one S. floridanus from the Capitan Mountains, two S. f. chapmani, and two S. audubonii (Genbank numbers HQ143448-HQ143464).

Average divergence of sequences between S. robustus and S. f. chapmani was low (1.64%), ranging from 1.09 to 2.21% (Table 1). Average divergence among all S. robustus was 0.76%, while within the Davis Mountains it was 0.15% and for Chisos Mountains it was 1.08%. Divergence between Davis and Chisos mountains averaged 0.63%. The individual from the Capitan Mountains differed from S. robustus by an average of 0.39%. Divergence of S. floridanus from S. audubonii and S. aquaticus averaged 14.30 and 11.48%, respectively.

Best-fit evolutionary model for the cytochrome b data was identified as HKY+[GAMMA] (Hasegawa et al., 1995), resulting from both hierarchical likelihood-ratio tests (hLRTs) and Akaike Information Criterion (minimum-theoretical-information criterion, AIC; Akaike, 1974). The dataset contained the following base frequencies: A = 0.2578, C = 0.3185, G = 0.1362, T = 0.2874. The transition-to-transversion ratio was 7.869. A single tree was produced in [PAUP.sup.*] (Swofford, 2001) under maximum-likelihood criteria (-Ln = 1,752.500; Fig. 1). Individuals of S. robustus and S. floridanus were combined into a single monophyletic clade supported by a bootstrap proportion of 100%. Sylvilagus audubonii formed a clade supported by a bootstrap proportion of 88%.

For the control region, we sequenced a total of 430 base pairs for 11 individuals from the Chisos Mountains; 11 from the Davis Mountains; four S. f. chapmani, one S. f. alacer, and one S. floridanus from the Capitan Mountains; one S. floridanus from the Sacramento Mountains; one S. floridanus from Otero County; and six S. audubonii (Genbank numbers HQ143412-HQ143447). A hypervariable region of 30 bases that could not be unambiguously aligned was excluded from the analyses, leaving a total of 400 characters for phylogenetic analysis.

When sequence of the control region was compared for all S. robustus, divergences of 0-4.43% were detected (Table 1). Within populations in the Davis and Chisos mountains, divergences were 0-2.30 and 4.72%, respectively, while a range of 0.50-4.17% was detected between these isolated populations. Divergence averaged 3.71% between S. robustus and the parapatric S. f. chapmani, and 3.57% between S. robustus and the allopatric S. f. alacer. Divergence averaged 14.58% between S. floridanus (chapmani and alacer combined) and S. audubonii.

[FIGURE 2 OMITTED]

For the control-region dataset, MODELTEST (Posada and Crandall, 1998) suggested the HKY+[GAMMA] model under the hLRTs and the TVM+[GAMMA] model under AIC. The dataset contained the following base frequencies: A = 0.3427, C = 0.0912, G = 0.2599, T = 0.3062. Transition-to-transversion ratio was 3.928. A single tree was produced in [PAUP.sup.*] under maximum-likelihood criteria (-Ln = 1,534.474; Fig. 2). As in the cytochrome b data, S. robustus and S. floridanus formed a monophyletic clade strongly supported by a bootstrap proportion of 100%. No distinct lineage was identified for either Davis or Chisos mountains. A monophyletic clade with significant bootstrap support was recovered including 19 S. robustus, one individual from the Capitan Mountains, and one individual from the Sacramento Mountains (Fig. 2). The six S. audubonii formed a monophyletic clade outside the S. robustus-S. floridanus clade.

Average measurements of adult S. robustus in our study (n = 18) were consistent with those reported by Schmidly (2004), although measurements of total length and length of tail were highly variable among individuals as were measurements of total length, length of tail, and length of ear in S. floridanus (n = 5). Average measurements and mass reported for S. robustus and S. floridanus in Texas (Schmidly, 2004) were as follows, respectively: total length, 416 and 418 mm; length of tail, 53 and 56 mm; length of hind foot, 98 and 92 mm; length of ear, 71 and 52 mm; mass, 1.3-1.8 and 1-2 kg. Average measurements and mass for S. robustus and S. floridanus in our study were, respectively: total length, 413 (SD = 34) and 369 mm (SD = 34); length of tail, 57 (SD = 10) and 41 mm (SD = 6); length of hind foot, 99 (SD = 4) and 87 mm (SD = 4); length of ear, 77 (SD = 3) and 66 (SD = 12); mass, 1.12 (n = 10; SD = 0.25) and 0.8 kg (n = 5; SD = 0.2). Unpaired t-tests revealed significant differences between adults of S. robustus and S. floridanus in our study for total length (P = 0.022), length of tail (P < 0.001), length of hind foot (P < 0.001), and mass (P = 0.016).

For each of the six cranial characters measured, the mean for S. robustus was greater than the mean for S. f. chapmani (Table 2), as was presented by Ruedas (1998). In contrast, discrete cranial characters examined in S. robustus and S. f. chapmani (Vestal, 2005) were incongruent with differences in characters proposed between the two taxa by Ruedas (1998). In specimens he examined, S. robustus had two distinct basisphenoid foramina, while S. f. chapmani had a single foramen. In our study not all skulls could be assessed for all characteristics, but four of 15 skulls of S. robustus had two distinct foramina, while the other 11 had a single basisphenoid foramen. All four skulls of S. f. chapmani had a single foramen. Ruedas (1998) stated that S. robustus lacked a tympanic process, whereas S. f. chapmani had a distinct process. In our study eight of 17 skulls of S. robustus lacked the process and the remaining nine had a distinct process. All four S. f. chapmani had a distinct process. Ruedas (1998) also reported that the mental foramen in S. robustus was twice as long as high and located on the dorsad aspect of the mandible, whereas in S. f. chapmani, this foramen was less than one-half as long as high and usually on the labial aspect of the mandible. In our study 16 of 19 S. robustus with intact mandibles had robustus-like mental foramina, as described by Ruedas (1998), whereas three had chapmani-like foramina. Three of the four S. f. chapmani had robustus-like foramina, while only one had a chapmani-like foramen.

DISCUSSION--Our genetic analyses identify Sylvilagus from the Chisos and Davis mountains as part of the floridanus group. Bradley and Baker (2001) calculated genetic distances based on data for cytochrome b sequences to determine boundaries in species of rodents and bats. Distances reported in our study between S. robustus and S. floridanus were congruent with values reported for intraspecific divergences by these authors. Further, interspecific divergences recovered between S. floridanus and S. audubonii (14.30%), S. floridanus and S. aquaticus (11.48%), and S. audubonii and S. aquaticus (11.14%) were markedly greater than those between S. robustus and S. floridanus. Halanych and Robinson (1999) reported similar divergences of cytochrome b between S. floridanus and S. audubonii (12.11%), S. floridanus and S. aquaticus (11.66%), and S. audubonii and S. aquaticus (7.77%). Overall, based on cytochrome b data, divergence observed between S. robustus and S. floridanus was unexpectedly low for a species-level comparison.

Litvaitis et al. (1997) examined genetic variation in New England (S. transitionalis), Appalachian (S. obscurus), and eastern (S. floridanus) cottontails in the northeastern United States and determined a difference in sequence of the control region of 16% between S. floridanus and S. transitionalis-S. obscurus groups. Additionally, their phylogenetic analysis revealed a clear separation of eastern and New England-Appalachian cottontails, but no distinct geographic pattern was seen between New England-Appalachian cottontails or among the 46 eastern cottontails examined from 10 northeastern states. Similarly, in the present study, no resolution was seen between populations of S. robustus in the Chisos and Davis mountains in either cytochrome b or control region. Divergences in the control region between the two isolated populations (0.50-4.17%) overlapped divergences within each population (Davis Mountains 0-2.30% and Chisos Mountains 0-4.72%). Branco et al. (2002) reported similar intrapopulational values for divergence of control region (0-7.72) among nine populations of the European rabbit (Oryctolagus cuniculus) on the Iberian Peninsula. Considering the comparatively large divergence values for cytochrome b and the control region previously reported between species of Sylvilagus (Litvaitis et al., 1997; Halanych and Robinson, 1999), the divergences we observed between robustus and floridanus are unlikely to warrant recognition of species status for robustus.

The low values for genetic divergence between populations in the Chisos and Davis mountains suggest that these populations have not been isolated a sufficient amount of time to cause detectable levels of genetic divergence. Considering the adjacent geographic position of the Davis and Chisos mountains, the lack of detectable divergence may be a result of the depression of the pinyon-oak-juniper zone [greater than or equal to] 790 m below its present lower limits at 1,400 m on the slopes of the Chisos Mountains, which occurred during the latest Wisconsin pluvial as recently as 11,500 years ago (Wells, 1974). This depression could have provided a forested corridor that probably extended over much of the Trans-Pecos (Schmidly, 1977) and provided an opportunity for gene flow among montane populations.

No conclusion can be made as to taxonomic identity of the three individuals from southern New Mexico (e.g., Capitan and Sacramento mountains and Otero County), because their divergences to robustus are comparable to those both within robustus and between robustus and floridanus. However, the specimen from Otero County was outside the entire robustus-floridanus group in the maximum-likelihood tree. Also, S. f. cognatus has been recorded from the northeastern slope of the Capitan Mountains (Hall and Kelson, 1951), so this may be the identity of the individual in this study from Capitan Mountains. Individuals from the Sacramento Mountains and Otero County were juveniles, which prevented an accurate comparison of cranial and external measurements of these specimens to those of S. robustus and S. floridanus.

Geographic variation in size of S. floridanus has been examined from three climatically distinct and disjunct regions within the range of the species: northeastern United States and southern Canada, southeastern United States, and southwestern United States and northern Mexico. Olcott and Barry (2000) examined 10 cranial measurements from 943 specimens from the three distinct regions and reported that variability in size was high within each of these regions, with size varying positively with elevation in the southwestern United States and northern Mexico. These results are consistent with our study, with high variability in size evident in S. floridanus, but consistently larger averages in cranial and external measurements in S. robustus that inhabit higher elevations in the region.

Although our sample of S. f. chapmani was small (n = 4), results of analysis of discrete cranial characters of S. robustus and S. f. chapmani were incongruent with differences proposed between the two taxa by Ruedas (1998). Of the 14 S. robustus examined for all three discrete characters, only one (from the Chisos Mountains) possessed robustus-like forms for all three characters, as described by Ruedas (1998). Of the four S. f. chapmani examined for all three discrete characters, only one possessed chapmani-like forms for all three characters, as described by Ruedas (1998). These discrete characters failed to separate S. robustus from S. f. chapmani. Incongruent morphological datasets, in addition to low values for genetic divergence between S. robustus and S. floridanus, suggest that populations of S. robustus have not been isolated a sufficient amount of time from S. floridanus to cause species-level differences.

Considering recent collections in the Davis Mountains, there appears to be a healthy population of S. f. robustus remaining there. At least a small population exists in the Chisos Mountains, evident by road-kills and sightings. Further sampling is needed in mountain ranges spanning the Trans-Pecos and surrounding areas to better elucidate distribution of subspecies of S. floridanus, as well as to determine size and conservation status of these populations.

We thank R. Skiles, L. Good, B. Alex, and J. Smith from Big Bend National Park; H. Garner, R. D. Bradley, R. J. Baker, and C. Jones at the Natural Science Research Laboratory at Texas Tech University; and J. Frey and D. Pollock at the Eastern New Mexico Natural History Museum. We also thank members of the field crew: C. E. Ebeling, A. C. Bishop, S. Clement, R. M. Rodriguez, S. A. Neiswenter, S. Stewart, S. Nalls, and S. Neis for their work in attempting to capture cottontails. G. Hernandez kindly assisted with translation of the abstract to Spanish.

Appendix

Species, collection locality, tissue, and catalog number for specimens used in analyses of cytochrome b (B), control region (C), and morphology (M). Specimens from the following institutions are included: Angelo State Natural History Collections (ASNHC, ASK), Natural Science Research Laboratory, Texas Tech University (TTU, TK), and Eastern New Mexico University (ENMU, ET).

Sylvilagus audubonii--USA: Texas: Brewster County, Black Gap Wildlife Management Area ASNHC9639 ASK3791 (B, C); Big Bend National Park, 5 miles N Persimmon Gap on Highway 385 ASNHC12943 ASK6404 (C). Jeff Davis County, Mount Livermore Preserve TTU101635 TK78836 (B), TTU101641 TK90039 (C). Reeves County, Sandia Springs TTU81104 TK84892 (C), TTU81105 TK84893 (C), TTU81106 TK84894 (C).

Sylvilagus floridanus alacer--USA: Texas: Nacogdoches County, Alazon Bayou Wildlife Management Area ASNHC12269 ASK5382 (C, M).

Sylvilagus floridanus chapmani--USA: Texas: Val Verde County, Devil's River State Natural Area ASNHC10795 ASK4764 (B, C, M), ASHNC11051 ASK4948 (B, C, M), ASNHC11064 ASK4950 (B, C, M). Tom Green County, San Angelo State Park ASNHC11974 ASK4937 (C, M).

Sylvilagus floridanus--USA: New Mexico: Lincoln County, Capitan Mountains, Head of Corral Canyon, elevation 1,479 m ENMU10664 ET462 (B, C, M). Otero County, Sacramento Mountains, James Canyon Campground, elevation 2,087 m ENMU11321 ET986 (C); Lower Three Mile Canyon burn ENMU11322 ET1048 (C).

Sylvilagus robustus--USA: Texas: Brewster County, Chisos Mountains, Big Bend National Park, Green Gulch Road, elevation 1,568 m ASNHC12239 ASK6046 (B, C, M); Big Bend National Park, Green Gulch Road, elevation 1,575 m ASNHC12941 ASK6216 (B, C, M); Big Bend National Park, Green Gulch Road, 13R 668269 3241909, elevation 1,482 m ASK6217 (B, C); Big Bend National Park, Green Gulch Road, 13R 666195 3239692, elevation 1,715 m ASNHC12940 ASK6268 (B, C, M); Big Bend National Park, Panther Pass, elevation 1,768 m ASNHC12942 ASK6331 (B, C, M); Big Bend National Park, Basin Road, near mile marker 4, elevation 1,638 m ASNHC12936 ASK6332 (B, C, M); Big Bend National Park, Basin Road ASNHC12938 ASK6333 (B, C, M); Big Bend National Park, Basin Road, elevation 1,737 m ASK6334 (B, C); Big Bend National Park, Basin Road, near mile marker 2 ASNHC12937 ASK6749 (C, M); Big Bend National Park, Basin Road, near mile marker 3 ASNHC12935 ASK6820 (C, M); Big Bend National Park, Basin Road, Chisos Basin Campground turnoff ASNHC12939 ASK6821 (C, M). Jeff Davis County, Davis Mountains State Park TTU7919 ASK3518 (B, C, M); Mount Livermore Preserve TTU101634 TK78795 (C), TTU101636 TK78862 (C, M), TTU101637 TK79064 (B, C, M), TTU101638 TK83585 (B, C, M), TTU101639 TK83586 (C, M), TTU101640 TK83587 (C, M), TTU81115 TK84903 (B, C, M), TTU81116 TK84904 (C, M), TTU81191 TK84983 (C, M), TTU101642 TK90053 (C, M).

Submitted 13 October 2009. Accepted 12 June 2011. Associate Editor was Marlis R. Douglas.

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AMY V. NALLS, * LOREN K. AMMERMAN, AND ROBERT C. DOWLER

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80525 (AVN)

Department of Biology, Angelo State University, San Angelo, TX 76909 (LKA, RCD)

* Correspondent: amy.nalls@colostate.edu
TABLE 1--Average Kimura two-parameter pair-wise mitochondrial sequence
divergence (%) for Sylvilagus.

                                                 Cytochrome b

              Comparison                   Mean (n)        Range

Sylvilagus robustus to S. floridanus      2.30 (12, 3)    1.09-3.99
Sylvilagus robustus to S. f. chapmani     1.64 (12, 2)    1.09-2.21
Sylvilagus robustus to S. f. mallurus     2.96 (12, 1)    2.69-3.99
Sylvilagus robustus to S. f. alacer           --              --
Within Sylvilagus f. chapmani             2.38 (2)            --
Within Sylvilagus robustus                0.76 (12)          0-2.20
Within Davis Mountains robustus           0.15 (4)           0-0.31
Within Chisos Mountains robustus          1.10 (8)           0-2.20
Davis to Chisos Mountains robustus        0.63               0-2.04
Capitan Mountains to robustus             0.39 (1, 12)       0-1.88
Sacramento Mountains to robustus              --              --
Otero County (canyon) to robustus             --              --
Sylvilagus floridanus to S. audubonii    14.30 (3, 3)    13.33-15.40
Sylvilagus floridanus to S. aquaticus    11.48 (3, 1)    11.00-11.77
Sylvilagus audubonii to S. aquaticus     11.14 (3, 1)     9.97-11.82

                                                Control region

              Comparison                   Mean (n)        Range

Sylvilagus robustus to S. floridanus      3.64 (22, 5)    1.79-5.86
Sylvilagus robustus to S. f. chapmani     3.71 (22, 4)    1.79-5.86
Sylvilagus robustus to S. f. mallurus         --              --
Sylvilagus robustus to S. f. alacer       3.57 (22, 1)    2.30-4.42
Within Sylvilagus f. chapmani             4.15 (4)        1.82-6.15
Within Sylvilagus robustus                1.41 (22)          0-4.43
Within Davis Mountains robustus           1.17 (11)          0-2.30
Within Chisos Mountains robustus          1.66 (11)          0-4.72
Davis to Chisos Mountains robustus        1.43            0.50-4.17
Capitan Mountains to robustus             0.81 (1, 12)       0-3.90
Sacramento Mountains to robustus          1.21 (1, 12)    0.25-4.17
Otero County (canyon) to robustus         2.90 (1, 12)    2.04-3.62
Sylvilagus floridanus to S. audubonii    14.94 (5, 6)    13.53-16.24
Sylvilagus floridanus to S. aquaticus         --              --
Sylvilagus audubonii to S. aquaticus          --              --

Table 2--Descriptive statistics for Sylvilagus robustus and S.
floridanus chapmani.

                                   Sylvilagus robustus

        Character             Mean      SD       Range (n)

Greatest length of skull     74.07     1.20    71.8-75.9 (11)
Condylopremaxillary length   65.74     1.02    64.3-67.0 (10)
Breadth of rostrum           19.58     0.84    18.3-20.9 (13)
Interbasioccipital length    20.56     0.75    19.4-21.5 (10)
Width of auditory bullae     13.01     1.04    12.0-16.3 (15)
Mastoid breadth              31.37     1.39    30.3-35.0 (10)

                             Sylvilagus floridanus chapmani

        Character             Mean      SD      Range (n)

Greatest length of skull     66.63     3.23    64.2-70.3 (3)
Condylopremaxillary length   59.43     2.64    57.1-63.2 (4)
Breadth of rostrum           17.23     1.15    16.3-18.9 (4)
Interbasioccipital length    19.23     0.91    18.4-20.5 (4)
Width of auditory bullae     11.46     0.26    11.1-11.7 (4)
Mastoid breadth               27.9     1.47    26.6-29.5 (3)
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